Project Summary In response to environmental cues, cells must selectively, rapidly, and robustly induce expression of specific genes in the context of two meters of ?chromatinized? DNA compacted within a complex, nuclear environment. Chromatin is composed of nucleosomes, or repeated units of DNA bound to an octamer of histone proteins, and chromatin is dynamically regulated by post-translational modifications to histones, chromatin modifying enzymes, and transcription factors. Important for moving towards a more complete understanding of chromatin regulation in the nuclear context, the recent description of extensive higher-order chromatin organization, and assays to resolve this at a genomic level, enables the study of chromatin architecture during these environmental responses. Key to this process of rapid and precise regulation of gene expression is what we will call ?signaling-to-chromatin? pathways, in which extracellular information is transmitted via kinase signaling cascades to select chromatin regions resulting in local transcription factor activity, alterations in chromatin state and structure, and transcription. However, surprisingly little is known of how activation of kinase cascades transmits information directly to histones for dynamic regulation of chromatin architecture and characteristics to affect transcription. We propose that the phosphorylation of both chromatin and transcription factors, and the downstream effects of these phosphorylation events (e.g. regulation of ?readers? and ?writers? of chromatin) may represent a rapid means of augmenting stimulation-induced transcription. Further elucidation of the role of histone phosphorylation and the role of chromatin-associated kinases is the subject of this proposal. Specifically, we will explore the role of H3S28 phosphorylation on chromatin architecture and accessibility (Aim 1), characterize the mechanism by which stimulation-induced H3.3S31 phosphorylation affects transcription (Aim 2a/b), and uncover signaling-to-chromatin events during the immune response by investigating a H3.3S31 kinase (Aim 2c). Importantly, the scope of transcription of inflammatory genes is critical to host survival during pathogen infection and, therefore, we propose that these selectively employed mechanisms for augmented transcription (e.g. coordinate regulation of transcription factors and chromatin by mitogen- and stress-activated protein kinases (MSKs) and additional kinases identified in this proposal) represent essential adaptations in response to pathogen pressure and are co-opted for diverse use in complex organisms. This is highlighted by the fact that these signaling pathways we propose to study are also relevant to many human diseases, including cancer. Utilizing our robust model system to uncover properties of chromatin regulation in response to external signals, our findings could provide insight into fundamental biological processes, disease mechanisms, and the therapeutic potential of targeting signaling-to-chromatin pathways.